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Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein.

Saheb E, Trzyna W, Maringer K, Bush J - Iran J Parasitol (2015 Apr-Jun)

Bottom Line: Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations.Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells.Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, College of Sciences, University of Baghdad, Baghdad, Iraq.

ABSTRACT

Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host's immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein.

Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr.

Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings.

Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii.

No MeSH data available.


Related in: MedlinePlus

Measurement of the chemotaxis activity in all cell lines using 5μM cAMP as chemo-attractant. After 4 hours, the Dictyostelium cells moved beyond one field of view on the agarose in a 60 × 15 mm Petri dish. The amoeboid control cells (WT and pDneo2a-GFP) clearly show trails toward the source of cAMP. Cells with Acmcp over-expression showed faster movement, while cells with Acmcp-dpr mutant scarcely moved towards the source of cAMP.
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Figure 5: Measurement of the chemotaxis activity in all cell lines using 5μM cAMP as chemo-attractant. After 4 hours, the Dictyostelium cells moved beyond one field of view on the agarose in a 60 × 15 mm Petri dish. The amoeboid control cells (WT and pDneo2a-GFP) clearly show trails toward the source of cAMP. Cells with Acmcp over-expression showed faster movement, while cells with Acmcp-dpr mutant scarcely moved towards the source of cAMP.

Mentions: Wild type, pDneo2a-GFP, Acmcp, and Acmcp-dpr cells were then sampled at 1, 2, 3, and 4 hours (Fig. 5), and representative photomicrographs were taken at each sample time point. Analysis of the data indicated that wild type AX4, pDneo2a-GFP, and Acmcp cells have normal chemotactic sensitivity to cAMP, which was characterized by their movement toward the source of cAMP. Cells that expressed Acmcp-dpr showed almost no movement towards the source of cAMP even after 4 hours of the experiment.


Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein.

Saheb E, Trzyna W, Maringer K, Bush J - Iran J Parasitol (2015 Apr-Jun)

Measurement of the chemotaxis activity in all cell lines using 5μM cAMP as chemo-attractant. After 4 hours, the Dictyostelium cells moved beyond one field of view on the agarose in a 60 × 15 mm Petri dish. The amoeboid control cells (WT and pDneo2a-GFP) clearly show trails toward the source of cAMP. Cells with Acmcp over-expression showed faster movement, while cells with Acmcp-dpr mutant scarcely moved towards the source of cAMP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522297&req=5

Figure 5: Measurement of the chemotaxis activity in all cell lines using 5μM cAMP as chemo-attractant. After 4 hours, the Dictyostelium cells moved beyond one field of view on the agarose in a 60 × 15 mm Petri dish. The amoeboid control cells (WT and pDneo2a-GFP) clearly show trails toward the source of cAMP. Cells with Acmcp over-expression showed faster movement, while cells with Acmcp-dpr mutant scarcely moved towards the source of cAMP.
Mentions: Wild type, pDneo2a-GFP, Acmcp, and Acmcp-dpr cells were then sampled at 1, 2, 3, and 4 hours (Fig. 5), and representative photomicrographs were taken at each sample time point. Analysis of the data indicated that wild type AX4, pDneo2a-GFP, and Acmcp cells have normal chemotactic sensitivity to cAMP, which was characterized by their movement toward the source of cAMP. Cells that expressed Acmcp-dpr showed almost no movement towards the source of cAMP even after 4 hours of the experiment.

Bottom Line: Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations.Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells.Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, College of Sciences, University of Baghdad, Baghdad, Iraq.

ABSTRACT

Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host's immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein.

Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr.

Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings.

Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii.

No MeSH data available.


Related in: MedlinePlus